Carbohydrates have demonstrated the capacity to stabilize membrane structure under the severe conditions of dehydration and freezing. This capacity has provided the opportunity to preserve biological materials in frozen or anhydrous state for prolonged periods. The amount of carbohydrate required for this effect varies, but often ranges from twenty to thirty percent (20-30%) of the sample weight. This is a significant amount; however, it is substantially lower than the amounts required of other commonly used cryoprotectants such as dimethyl sulfoxide, urea, glycerol, sucrose and ethylene glycol (Lovelock, J. E., Biochimica et Biophysica Acta 11, 28-36 (1979)). Additionally, use of carbohydrates avoids the deleterious effects on membrane structure which are often encountered when the other above-mentioned reagents are used.
Interest in carbohydrates as cryoprotective agents was spurred by the discovery over ten years ago that certain organisms capable of surviving in a dehydrated state for many years produced large amounts of trehalose, a non-reducing disaccharide of glucose (Madin, K. A. C., and Crowe, J. H., Journal of Experimental Zoology, 193, 335-342 (1975), and Loomis, S. H., O'Dell, S. J., and Crowe, J. H., Journal of Experimental Zoology, 211, 321-330 (1980)). Trehalose was subsequently shown to be three times more effective than sucrose and several more times effective than other cryoprotectants in preserving membrane structure and function in frozen and dehydrated systems (Crowe, L. M., Mouradian, R., Crowe, J. H., Jackson, S. A., and Womersely, C., Biochimica et Biophysica Acta, 769, 114-150 (1984)). It is also known that galactose can induce similar effects on membrane behavior.
All of the known cryoprotective agents are currently used in solution, to which is added the biological materials desired to be preserved. This approach requires relatively high concentrations of free carbohydrate or other agents in order to effectively reduce membrane leakage, thereby preserving the biological materials upon freezing and thawing. There has long been a need in the art for a method of treating cells, vesicles, membranes, and other biological and synthetic materials so that they may be stored in a frozen or anhydrous state for periods beyond that which is now possible.